RTN4在胃癌发病机制中的作用

目的 了解RTN4蛋白在胃癌发病过程中的作用,为揭示胃癌发病的分子机制提供实验资料。方法 将pIRESneo3-RTN4真核表达载体转染胃粘膜上皮GES1细胞,建立RTN4高表达的GES1-RTN4细胞系;通过细胞生长曲线、平皿克隆与软琼脂集落形成实验、流式细胞仪,观察RTN4高表达对GES1细胞生长与增殖、周期与凋亡的影响;利用Western-blot检测GES1-RTN4细胞中IκBα蛋白的表达。结果 GES1-RTN4细胞的生长速度较GES1和GES1-pIRESneo3细胞加快,统计学意义显著（P＜0.01）;GES1-RTN4细胞较GES1和GES1-pIRESneo3细胞克隆体积大、数目多,统计学意义显著（P＜0.01）;GES1-RTN4细胞中S期细胞比例较GES1和GES1-pIRESneo3细胞增加,细胞增殖指数增加,细胞凋亡率降低,统计学意义显著（P＜0.01）;GES1-RTN4细胞中IκBα蛋白表达较GES1和GES1-pIRESneo3细胞减少,统计学意义显著（P＜0.01）。结论 RTN4通过活化NF-κB信号通路,提高细胞增殖指数,抑制细胞凋亡,促进GES1细胞的增殖。     

异鼠李素对肺癌的影响及其抗肿瘤机制的初步探讨

目的：探讨异鼠李素体外对人肺癌细胞A549、体内Lewis肺癌增殖的影响及初步研究其抗肿瘤机制。方法：以不同浓度异鼠李素；UUA体外培养的A549细胞中。用MTT比色法，细胞计数法。细胞生长曲线的测定、细胞形态学变化、克隆形成,^3H-TdR掺入实验等观察测定体外抗肿瘤活性，用流式细胞仪、基因组DNA片段分析，彗星电泳、免疫组化等观察其对肿瘤细胞凋亡的诱导作用；     

人子宫内膜中存在一群具有克隆增殖能力的细胞

背景：克隆形成能力是干细胞的重要特性,多项研究证明,子宫内膜在起源上具有单克隆性。 目的：通过体外培养,检测人子宫内膜细胞克隆形成能力。 方法：采用机械和酶消化相结合的方法分离出人子宫内膜细胞,培养腺上皮细胞和基质细胞,观察细胞形态及生长特性并检测波形蛋白和角蛋白的表达,采用流式细胞仪对细胞进行CD133,CD34,CD45,CD90,CD73及CD29表型鉴定;观察细胞克隆生长特点、测量细胞克隆大小和计算克隆形成率。同时取人子宫肌细胞培养作为对照。 结果与结论：经过12 d的原代培养后,腺上皮细胞和基质细胞均可形成大、小两种克隆。角蛋白表达阳性为腺上皮细胞,波形蛋白表达阳性为基质细胞。流式细胞术检测子宫内膜细胞表达CD90、CD29、CD73等表面标记,不表达CD34、CD45,CD133。腺上皮细胞、基质细胞大克隆和小克隆大小及形成率比较,差异均有显著性意义(P < 0.05);人子宫肌细胞用有限稀释法原代培养无细胞克隆生长。说明子宫内膜细胞在体外培养中,腺上皮细胞和基质细胞大克隆增殖能力均高于小克隆。这些细胞可能是子宫内膜干细胞。     

乙醛脱氢酶活性检测子宫内膜干细胞

背景：人子宫内膜中存在干细胞特性的细胞,但特异性标志物尚未找到,导致此类细胞很难被分离。 目的：分析乙醛脱氢酶(aldehyde dehydrogenase,ALDH)活性检测能否分选出子宫内膜干细胞。 方法：采用机械和酶消化相结合方法分离人子宫内膜细胞,用两次滤网方法分离出基质细胞和上皮细胞,根据乙醛脱氢酶活性采用流式细胞仪分选出ALDHhigh细胞和ALDHlow细胞,用有限稀释法培养细胞,观察细胞形态及生长特性;采用流式细胞仪鉴定细胞c-kit,CD90,CD73及CD29的表达。 结果与结论：基质细胞中ALDHhigh细胞占(1.88±0.17)%,经过15 d的原代培养后,ALDHlow细胞克隆形成率达(1.06±0.34)%,ALDHhigh细胞克隆形成率达(4.50±0.82)%,ALDHhigh细胞集落形成率较ALDHlow细胞高(P < 0.05),同时ALDHhigh细胞大克隆数较ALDHlow高(P < 0.05);上皮细胞中未发现ALDHhigh细胞及克隆形成。子宫内膜集落形成细胞表现出c-kit、CD29、CD90、CD73阳性。说明ALDH活性检测对子宫内膜干细胞有一定的分选作用。     

牛蒡子苷诱导人肝癌HepG2细胞凋亡的实验研究

目的：通过牛蒡子苷对肿瘤细胞的体外实验，探讨牛蒡子苷是否对人肝癌细胞（HepG2）具有生长抑制及诱导凋亡作用。方法：HepG2细胞培养液中加入不同浓度的牛蒡子苷，培养72 h，观查HepG2细胞的增殖率；倒置显微镜及投射电子显微镜观察HepG2细胞的形态变化；流式细胞仪（FCM）检测HepG2细胞周期及凋亡率；TUNEL法检测HepG2细胞凋亡率；免疫细胞化学检测HepG2细胞的增殖凋亡相关蛋白(Bax、Bcl-2)表达。结果： 牛蒡子苷抑制HepG2细胞生长；阻滞HepG2细胞周期于G0/G1期并诱导细胞凋亡(P＜0.05)；通过下调Bcl-2蛋白表达（P＜0.01），上调Bax蛋白表达（P＜0.05）诱导HepG2细胞凋亡。结论： 牛蒡子苷对体外培养的HepG2细胞生长有抑制作用,其作用机制之一是通过诱导细胞凋亡。     

树突状细胞体外定向诱导扩增及其分离纯化

目的：探讨定向诱导及纯化树突状细胞（ＤＣ）的方法，为进一步研究树突状细胞的功能、特性及临床应用提供技术方法。方法：利用免疫磁珠江（ＭＡＣＳ）分离纯化脐血ＣＤ３４细胞，在液体培养体系中加入ＦＬ、ＧＭ－ＣＳＦ、ＴＮＦα、ＩＬ－４ｅｙ ＳＣＦ，培养１２ｄ后光镜检测细胞形态及流式细胞仪分析表达标志；利用抗估状细胞单克隆抗体（Ｘ－１１）及免疫磁珠分离纯化树突状细胞，流式细胞仪检测纯化后ＤＣ的纯度。结果：经体     

PML—RARα基因在早幼粒细胞性白血病发病中的作用研究

目的：通过封闭ＰＭＬ－ＲＡＲα融合基因对ＮＢ４细胞生长分化作用的影响，探讨ＰＭＬ－ＲＡＲα融合基因与ＡＰＬ发病的关系。方法：用反义核酸封闭ＰＭＬ－ＲＡＲα融合基因的表达。ＮＢ４细胞的生长、分化及功能，通过细胞生长曲线、形态学、细胞表面标志及ＮＢＴ还原试验判定；细胞周期应用流式细胞仪分析。结果：ＰＭＬ－ＲＡＲα融合基因的封闭能够抑制ＮＢ４细胞生长，促进其分化成熟，同时可降低Ｓ期细胞的百分比（由５６％降至３７％）。结论：急性早幼粒细胞性白血病（ＡＰＬ）特征性的染色体易位ｔ（１５；１７）形成的ＰＭＬ－ＲＡＲα融合基因，是ＡＰＬ发病的分子基础。     

高低转移肺腺癌细胞系中肿瘤抑制基因p16、p21和p53表达研究

目的 探讨肿瘤抑制基因对肺腺癌细胞生长的抑制作用。方法 利用FuGene转染方式分别将p21和p16基因的表达质粒转入－对肺腺癌细胞系Anip973和AGZY83－a中,同时用含野生型p53 基因的腺病毒感染p16基因转染前后的这一对细胞系。对P16和P21蛋白过表达的细胞系进行了细胞生长曲线、克隆形成率、原位末端标记分析和流式细胞仪分析。结果 p16基因的过表达只能使细胞系的G1期细胞比例提高,但细胞生长曲线,克隆形成率均未出现改变,未检测到凋亡信号。P21蛋白过表达的一对细胞系细胞生长曲线斜率降低,克隆形成能力下降,并出现明显的G1期阻滞,但未检测到凋亡信号。p53基因感染AGZY83－a,Anip973及经过p16基因转染的细胞AGZY83－ap16和Anip973p16后呈现时间依赖性表达,细胞生长曲线和四唑盐比色法分析提高,野生型p53基因的大量表达明显抑制以上4种细胞的生长,Anip973和Anip973p16的生长抑制率高于AGZY83－a和AGZY83－ap16;Anip973p16和AGZY83-ap16的生长抑制率高于Anip973和AAGZY83－a。这4种细胞在感染p53后出现典型的凋亡信号。结论p16基因的过表达并不能抑制细胞系的生长,而p21基因的过表达通过G1期阻滞抑制这1对肺腺癌细胞的生长;野生型p53基因在AGZY83－a和Anip973中高效表达可产生明显的细胞生长抑制效应;野生型p53基因对肺腺癌高转移细胞系Anip973抑制作用更为明显。     

人脂肪干细胞的分离培养与生物学特性

背景：高效、稳定地获得大量较高纯度的人脂肪干细胞,是其在组织工程学及再生医学中广泛应用的基础和前提。 目的：探索体外培养脂肪干细胞的适宜条件,从而提高其增殖能力。 方法：采用胶原酶消化的方法,从人腹部皮下块状脂肪中分离出脂肪干细胞,经贴壁筛选法纯化细胞、低糖培养基体外培养扩增。Giesam染色后观察细胞形态;绘制细胞生长曲线并进行细胞周期分析,观察分析传代后染色体核型改变;选取第3代脂肪干细胞做流式细胞鉴定、EdU细胞增殖能力检测以及克隆形成实验。 结果与结论：分离培养的原代脂肪干细胞形态不一,经传代后的细胞形态趋于长梭形,排列紧密呈漩涡状生长。细胞生长曲线呈S形,细胞周期分析及EdU掺入法结果显示体外培养的脂肪干细胞具有较强的增殖能力。经染色体核型分析结果提示,体外培养不会引起脂肪干细胞的核型改变。第3代脂肪干细胞经流式细胞仪检测CD29,CD44,CD90,CD105均呈阳性表达,而CD34和CD45呈阴性;克隆形成率为8.8%;在一定的诱导条件下,脂肪干细胞能够向脂肪细胞及成骨细胞分化。结果说明采用胶原酶消化法可成功分离培养人脂肪干细胞,且具有较强的增殖能力。     

NDRG2对肝癌细胞HepG2凋亡的诱导作用

目的：探讨NDRG2对肝癌细胞HepG2凋亡的诱导作用。方法：用RT-PCR获取NDRG2基因。测序判断正确后，将其克隆入含绿色荧光蛋白(GFP)基因的真核表达载体pIRES2-EGFP中，并转染NDRG2表达阴性的HepG2细胞。用光镜观察转染细胞的形态变化；荧光显微镜观察NDRG2蛋白的定位；流式细胞仪分析细胞周期的改变；透射电镜进一步观察细胞超微结构的改变。结果：获得了NDRG2基因，成功地构建了pIRES2-EGFP-NDRG2真核表达载体。转染HepG2细胞后，细胞生长受抑，结构破坏并死亡。荧光显微镜观察到NDRG2在胞质中表达，流式细胞仪检测出现G1期阻滞和凋亡峰，透射电镜观察到典型的细胞凋亡特征。结论：NDRG2基因具有诱导HepG2细胞凋亡的作用，其机制有待进一步研究。     

Effect of Helicobacter pylori on gastric epithelial cell migration and proliferation in vitro: role of VacA and CagA.

Helicobacter pylori infection is associated with inflammation of the gastric mucosa and with gastric mucosal damage. In this study, we sought to test the hypothesis that two H. pylori virulence factors (VacA and CagA) impair gastric epithelial cell migration and proliferation, the main processes involved in gastric mucosal healing in vivo. Human gastric epithelial cells (MKN 28) were incubated with undialyzed or dialyzed broth culture filtrates from wild-type H. pylori strains or isogenic mutants defective in production of VacA, CagA, or both products. We found that (i) VacA specifically inhibited cell proliferation without affecting cell migration, (ii) CagA exerted no effect on either cell migration or proliferation, and (iii) undialyzed H. pylori broth culture filtrates inhibited both cell migration and proliferation through a VacA- and CagA-independent mechanism. These findings demonstrate that, in addition to damaging the gastric mucosa, H. pylori products may also impair physiological processes required for mucosal repair.     

Topographical localisation of cagA positive and cagA negative Helicobacter pylori strains in the gastric mucosa; an in situ hybridisation study

BACKGROUND: The cagA gene is a marker for the presence of the cag pathogenicity island, and the presence of cagA positive strains of Helicobacter pylori can identify individuals with a higher risk of developing gastrointestinal diseases. AIMS: To study the interaction between H. pylori cagA(+) and cagA(-) strains and the gastric mucosa. METHODS: Patients with H. pylori associated gastritis and peptic ulcers were studied. Biopsies were obtained from the antrum, corpus, fundus, and incisura for H pylori culture, and for in situ hybridisation studies. From each biopsy, multiple single H. pylori colonies were isolated and propagated for DNA isolation, and cagA was detected by the polymerase chain reaction (PCR). For in situ detection of H. pylori an oligonucleotide specific for an H. pylori common antigen and an oligonucleotide specific for cagA were used as probes. Biotinylated probes were incubated with biopsy sections, developed with streptavidin-horseradish peroxidase, and amplified with the tyramide system. RESULTS: PCR results for cagA in isolated colonies confirmed the in situ hydridisation studies. In situ hybridisation identified cagA(+) bacteria in patients with cagA(+) isolates; cagA(-) bacteria in patients with cagA(-) isolates, and cagA(+) and cagA (-) bacteria in patients with both cagA(+) and cagA(-) isolates. CagA(-) bacteria usually colonised the mucous gel or the apical epithelial surface, whereas cagA(+) bacteria colonised the immediate vicinity of epithelial cells or the intercellular spaces. CONCLUSIONS: These results document a different in vivo interaction between H. pylori cagA(+) or cagA(-) strains and the gastric mucosa.     

Effect of inhibition of extracellular signal-regulated kinase 1 and 2 pathway on apoptosis and bcl-2 expression in Helicobacter pylori-infected AGS cells

Helicobacter pylori induces activation of mitogen-activated protein kinases (MAPKs). However, its effect on H. pylori-induced apoptosis has not been evaluated. Thus, we examined whether H. pylori-induced extracellular signal-regulated kinase 1 and 2 (ERK1/2) and p38 MAPK activation affects gastric epithelial cell apoptosis and bcl-2 family gene expression, especially in relation to the cagA status of an H. pylori strain. In flow cytometric and oligonucleosome-bound DNA enzyme-linked immunosorbent assay analyses, infection with cagA(+) H. pylori strains induced gastric cancer cell apoptosis in AGS cells more prominently than infection with cagA mutants. Activation of ERK1/2 and p38 MAPKs was also more prominent in cagA(+) strains. Pretreatment with a MEK inhibitor (PD98059) inhibited ERK1/2 activation and increased H. pylori-induced apoptosis significantly. This increased apoptosis was accompanied by decreased antiapoptotic bcl-2 mRNA expression among bcl-2-related genes (bcl-2, bax, bak, mcl-1, and bcl-X(L/S)), and the effect was also more prominent in the cagA(+) strains. However, the alteration of bcl-2 gene expression was not accompanied by protein level changes. Inhibition of p38 using specific inhibitor SB203580 decreased H. pylori-induced apoptosis but resulted in little alteration of bcl-2-related gene expression. In conclusion, H. pylori-induced ERK1/2 activation, especially by the cagA(+) H. pylori strain, may play a protective role against gastric epithelial cell apoptosis partially through maintenance of bcl-2 gene expression.     

UreC PCR based diagnosis of Helicobacter pylori infection and detection of cag A gene in gastric biopsies

Polymerase chain reaction assay using ureC gene specific primers for the detection of Helicobacter pylori in gastric biopsy specimens from 116 dyspeptic patients was compared with other routine invasive diagnostic methods (culture, rapid urease test [RUT] and histology). In parallel, gastric biospy specimens from 54 patients and their corresponding Helicobacter pylori isolates were subjected to PCR with cagA targeting primers using standard protocols. Helicobacter pylori were detected in 53%, 43%, 48% and 50% of patients by PCR, RUT, culture and histological examination respectively. Based on histology and culture positive and at least three test positive result, 44 (37%), 46 (39%) and 26 (22%), and 56 (48%), 52 (44%) and 8 (6%) patients were classified as Helicobacter pylori positive, negative and indeterminate respectively. The sensitivity and specificity of PCR assay was the highest-95% and 100% when compared with both culture and histology positive, and at least any three positive results respectively. The result of cagA positivity in 54 gastric biopsy specimens and their corresponding Helicobacter pylori isolates were identical; 18 of 20 (90%) duodenal ulcer patients and 23 of 28 (82%) patients with chronic gastritis and 2 (40%) of 5 patients with portal hypertension and one gastric biopsy specimens from gastric cancer patients were found to be cagA positive. PCR-based method to detect Helicobacter pylori and the virulence gene cag A directly from gastric biopsy specimens appears to be promising and can curtail the lengthy process of culture-based approaches. The procedure proved to be rapid and reliable and could be utilized for diagnostic purposes.     

Induction of interleukin-8 secretion from gastric epithelial cells by a cagA negative isogenic mutant of Helicobacter pylori.

The ability of Helicobacter pylori strains to induce interleukin-8 (IL-8) gene expression and protein secretion from gastric epithelial cell lines in vitro is variable. This cellular response is associated with bacterial expression of the CagA protein present in type I H pylori strains. To determine the role of CagA in this host cell response, an isogenic cagA negative mutant, N6.XA3, was constructed. The cagA negative isogenic mutant and the wild-type parental cagA positive strain, N6, were cocultured with AGS, ST-42 and KATO-3 gastric epithelial cell lines and secreted interleukin-8 assayed by enzyme linked immunosorbent assay. In all three cell lines there was no significant difference in the IL-8 secretion induced by the cagA negative isogenic mutant, N6.XA3, and the wild-type parent strain, N6. These studies show that CagA is not the inducer of IL-8 secretion from gastric epithelial cells. As all wild-type CagA positive strains studied to date induce IL-8, the bacterial factor(s) inducing this inflammatory response is closely associated with the expression of CagA.     

Virulence markers of Helicobacter pylori in patients with diarrhoea-dominant irritable bowel syndrome

Recent studies suggest that irritable bowel syndrome (IBS) is associated with low-grade inflammation. This study aims to determine the distribution of Helicobacter pylori cytotoxin-associated gene A (cagA) and vacuolating cytotoxin A (vacA) alleles (e.g., s1 and s2) in patients with diarrhoea-dominant IBS (IBS-D) as the latter causes vacuolation in colonic epithelial cells in vitro. One hundred and seventy patients meeting Rome III criteria for IBS-D (mean age: 40 +/- 15 years) were enrolled. Gastric biopsy was assessed histologically and DNA extraction was performed by polymerase chain reaction (PCR) for H. pylori genus 16S ribosomal DNA (16S rDNA), cagA and vacA allele s1 and s2. There was no age- or gender-related difference in H. pylori positivity in IBS-D compared to the control group. H. pylori was positive in 116 (68%) with IBS-D compared to 88 (55%) in the control group (P=0.01). cagA was positive in 73 (63%) with IBS-D compared to 42 (48%) in the control group (P=0.03). vacA s1 was positive in 61 (53%) with IBS-D compared to 32 (36%) in the control group (P=0.02). cagA s1 was positive in 39 (34%) with IBS-D compared to 13 (15%) in the control group (P=0.002).     

The significance of cagA and vacA subtypes of Helicobacter pylori in the pathogenesis of inflammation and peptic ulceration

AIMS: To assess the significance of cagA and vacA subtypes of Helicobacter pylori in relation to inflammation and density of bacterial colonisation in vivo within a dyspeptic UK population. METHODS: Dyspeptic patients who were Helicobacter pylori positive had antral samples taken for histology and culture. Gastroduodenal pathology was noted. The grade of bacterial density and inflammation was assessed using the Sydney system. Bacterial DNA was extracted and the vacA alleles and the cagA/gene typed using PCR. RESULTS: 120 patients were studied. There was high rate of cagA positive strains in this population. Bacterial density did not correlate with the presence of peptic ulceration. There was a significant association between cagA positive strains and increased inflammation and bacterial density. The vacA s1 type independently correlated with extensive chronic inflammation but there was no association with bacterial density. The vacA m type did not correlate with extent of inflammation or bacterial density. CONCLUSIONS: The results suggest that cagA is important in the pathogenesis of inflammation and peptic ulceration. These findings are in keeping with the hypothesis that cagA acts as a marker for a cag pathogenicity island which encodes several genes involved in inflammation. The vacA s1 allele correlates with inflammation independently of cagA, possibly through its enhanced ability to produce the vacuolating cytotoxin.     

Effect of Helicobacter pylori on polymorphonuclear leukocyte migration across polarized T84 epithelial cell monolayers: role of vacuolating toxin VacA and cag pathogenicity island

Helicobacter pylori infection can induce polymorphonuclear leukocyte (PMNL) infiltration of the gastric mucosa, which characterizes acute chronic gastritis. The mechanisms underlying this process are poorly documented. The lack of an in vitro model has considerably impaired the study of transepithelial migration of PMNL induced by H. pylori. In the present work, we used confluent polarized monolayers of the human intestinal cell line T84 grown on permeable filters to analyze the epithelial PMNL response induced by broth culture filtrates (BCFs) and bacterial suspensions from different strains of H. pylori. We have evaluated the role of the vacuolating cytotoxin VacA and of the cag pathogenicity island (PAI) of H. pylori in PMNL migration via their effects on T84 epithelial cells. We noted no difference in the rates of PMNL transepithelial migration after epithelial preincubation with bacterial suspensions or with BCFs of VacA-negative or VacA-positive H. pylori strains. In contrast, PMNL transepithelial migration was induced after incubation of the T84 cells with cag PAI-positive and cagE-positive H. pylori strains. Finally, PMNL migration was correlated with a basolateral secretion of interleukin-8 by T84 cells, thus creating a subepithelial chemotactic gradient for PMNL. These data provide evidence that the vacuolating cytotoxin VacA is not involved in PMNL transepithelial migration and that the cag PAI, with a pivotal role for the cagE gene, provokes a transcellular signal across T84 monolayers, inducing a subepithelial PMNL response.     

Clinical and histological associations of cagA and vacA genotypes in Helicobacter pylori gastritis.

AIMS: To determine the relation among the cytotoxin associated gene (cagA) and vacuolating cytotoxin gene (vacA) status of Helicobacter pylori isolates, the associated clinical diseases, and the severity and pattern of chronic gastritis. METHODS: Helicobacter pylori was cultured from gastric biopsies obtained from dyspeptic patients. DNA was extracted from the isolates and the cagA and vacA status determined by the polymerase chain reaction (PCR). The prevalence of the different cagA and vacA genotypes in three clinical groups, duodenal ulcer, gastric ulcer, and non-ulcer dyspepsia was compared. The histological features in sections from two antral and two corpus biopsies were graded by one blinded observer. The grades were compared with age and sex matched groups with different cagA and vacA genotypes, and with duodenal ulcers, or non-ulcer dyspepsia. RESULTS: Isolates from 161 patients were included. One hundred and nine (68%) harboured a cagA+ strain and 143 (89%) harboured a vacA s1 strain. The prevalence of cagA+ strains in duodenal ulcer patients (94%) was highly significantly greater than in those with non-ulcer dyspepsia (56%). However, of the patients infected with a cagA+ strain, almost equal numbers had non-ulcer dyspepsia or peptic ulceration. Chronic inflammation, polymorph activity, surface epithelial degeneration, atrophy, and intestinal metaplasia were all significantly more severe in the cagA+ than in the cagA- group, whereas only corpus epithelial degeneration was significantly more severe in the vacA s1 group compared with the vacA s2 group. Patients infected with cagA+ strains were almost four times more likely to have antral intestinal metaplasia than cagA- patients. An antral predominant gastritis was present in duodenal ulcer patients compared with matched non-ulcer dyspepsia patients, but this was not attributable to cagA or vacA status. CONCLUSIONS: Helicobacter pylori strains showing cagA positively and the vacA s1 genotype are associated with more severe gastritis but these virulence factors do not appear to determine the overall pattern. The pattern is closely linked to clinical disease. Therefore, it is likely that the nature of the disease complicating chronic infection is determined by host and environmental factors, while bacterial factors determine the magnitude of the risk of developing such disease.     

Diagnosis of Helicobacter pylori infection by PCR: comparison with other invasive techniques and detection of cagA gene in gastric biopsy specimens.

A PCR assay for the detection of Helicobacter pylori in gastric biopsy specimens with specific primers for ureC gene amplification (herein referred to as ureC PCR) was compared with other routine invasive methods (culture, the rapid-urease test, and Giemsa staining of histological sections) with samples from a group of 104 consecutive dyspeptic patients. Bacteria were found in 40 (38.5%), 38 (36.5%), 36 (34.6%), and 35 (33.7%) of the patients by ureC PCR, culture, the rapid-urease test, and Giemsa stain, respectively. Sixty-three patients had negative cultures, negative histological examinations, and negative rapid-urease test results, and 61 of these patients were also negative by ureC PCR. ureC PCR detected H. pylori in two culture-negative patients. In parallel, a PCR-based assay to detect the H. pylori cytotoxin-associated antigen (cagA) gene, a putative virulence gene, was also developed. To assess the likelihood of detection of H. pylori genes directly from gastric biopsy samples and from the corresponding H. pylori isolates, specimens from 31 patients were subjected to PCR with ureC- and cagA-targeting primers. All 31 biopsy specimens and the corresponding H. pylori isolates were positive in the ureC PCR. H. pylori strains that were cagA positive also gave positive cagA PCR fragments with biopsy specimens from the same patients. All ureC PCR-positive patients were examined; biopsy specimens from 10 of 11 (91.7%) duodenal ulcer patients harbored H. pylori cagA-positive strains, whereas 19 of26 (73%) of those from patients with chronic gastritis only were found to be cagA positive.(ABSTRACT TRUNCATED AT 250 WORDS)